Toxic and metabolic insults such as oxygen and glucose deprivation generated by trauma, hypoxia-ischemia, and seizures are extremely harmful for the central nervous system (CNS). Besides this, aging makes the CNS vulnerable to neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. In these settings, hippocampal and neocortical neurons degeneration is observed. To understand the mechanisms of neuronal dysfunction and degeneration, animal and cell culture model systems are widely used. While animal models can be used to study many aspects of neuronal development and degeneration [1], the molecular, biochemical, and structural determinations are extremely complex or not possible at all, due to the inherent complexity of the nervous system tissue. To better approach these studies in vitro systems provide homogenous populations of cells undergoing synchronous and uniform changes.

The first in vitro system for the culture of primary neurons was developed by Banker and Cowan [2-4]. It consisted of cultures of hippocampal neurons isolated form 18 day old rat fetuses co-cultured with tissue or hippocampal explants and survived only 2 weeks. Since then, the culture systems for hippocampal and cortical neurons, as well as neuron cell lines, have improved dramatically [5-11] and are widely used in diverse studies.

The development of serum-free media for cell culture [12] represented a major advantage for the culture of neuronal cell lines. Following this, a serum free medium was developed that allowed the culture of hippocampal and cortical neurons [9]. This medium is nowadays widely used for the culture of primary neurons and neuronal cell lines, and is composed of neurobasal medium (NB) supplemented with B-27 [6,8,9]. It was derived from Dulbecco's Modified Eagle's Medium, and was optimized for cell survival and neurite outgrowth of hippocampal neurons with an almost complete absence of glial cells [6,8,9]. The same was also observed in cultures of primary cortical neurons [5]. Primary neuronal cultures provide a defined environment where neurons can be studied in isolation from other cell types that can modify neuronal responses directly or indirectly. The B-27 supplemented NB is useful for studies of neurotoxicology, electrophysiology, gene expression, pharmacology, development, and growth factor effects [9,13]. Thus, primary cultures of cortical neurons are a powerful tool for studying cellular and molecular mechanisms of neuronal development, aging, and death [14]. However the changes that occur in neurons during culture in relation to cell differentiation and medium composition have not been thoroughly characterized, and this could have a large impact specially in the field of neurotoxicology where many times chemicals are dissolved previously in fresh medium and then added to the cell cultures [15]. Using primary neuron cultures in B-27 supplemented NB medium, and as an approach to determine the effect of time in culture and variations in medium composition due to cell metabolism on primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary neurons between 7 and 10 days in vitro (DIV) and after medium change.

Primary cultures of cortical neurons

Swiss mice were used to obtain primary cultures of cortical neurons. Primary cortical neurons were obtained from embryonic day 16-18 Swiss mice. All protocols were approved by the University of Santiago de Compostela Institutional Animal Care and Use Committee. Briefly, cerebral cortices were removed and dissociated by mild trypsinization, followed by mechanical trituration in a DNase solution (0.004% w/v) containing a soybean trypsin inhibitor (0.05% w/v). The cells were suspended in Neurobasal medium supplemented with 1% B-27 supplement (Invitrogen), 5 mM L-glutamine, and 1% penicillin/ streptomycin. The cell suspension was seeded in 12 well plates precoated with poly-D-lysine, and the cell culture was kept in a 95% air and 5% CO₂ atmosphere at 37°C. Culture medium was replaced every 3-4 days. Neurons were employed between 7 and 10 days in culture.

RNA extraction, purification, quantification and integrity determination

The Aurum^™ Total RNA Mini Kit (BIO-RAD) was used to obtain RNA for microarray assays, following the manufacturer instructions. RNA concentration was determined with a NanoDrop 2000 (THERMO SCIENTIFIC) and integrity was confirmed with a bioanalyzer 2100 (AGILENT) using the kit RNA 6000 nano reagents (AGILENT).

Microarray assay and analysis

To obtain fluorescently labeled RNA for microarray hybridization we used the Low RNA Input Linear Amp Kit (Agilent Technologies) and the Quick-Amp Labeling Kit (Agilent Technologies) and followed the manufacturer instructions. Briefly, Cyanine-3 (Cy3) labeled cRNA was prepared from 1 μg RNA using the Quick-Amp Kit (Agilent Technologies), followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with a Nanodrop (THERMO SCIENTIFIC) spectrophotometer. Cy3-labelled cRNA (1.5 μg; specific activity >9.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 10 X blocking agent (Agilent Technologies), nuclease-free water, and 25 X fragmentation buffer (Agilent Technologies). On completion of the fragmentation reaction, 25 μl of 2 X GE hybridization buffer (HI-RPM0) (Agilent Technologies) was added to the fragmentation mixture which was hybridized to 8X60K SurePrint G3 Agilent Whole Mouse Genome Oligo Microarrays (G4852A) containing 39430 features, for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 and 1 minute at 37°C GE Wash Buffer 2 (Agilent Technologies). After washing, the microarrays were scanned with a MS200 scanner (ROCHE) using one color scan setting (scan resolution 2 μm). Each slide, containing 8 arrays, was scanned using a single image mode without auto gain and with 100% gain for acquisition. The scanned images were extracted with Feature Extraction Software 11.5.1.1 (Agilent Technologies) using default parameters (protocol GE1-QCMT_11.5 and Grid: 028005_D_F20131202).

After extraction, differential gene expression as a consequence of time in culture and response to medium change was determined using GeneSpring software (Agilent Technologies). Gene lists were analyzed for differentially expressed (DE) genes using oneway ANOVA set at p<0.05, with a fold change cutoff =2. Data mining for significant altered pathways and ontological categories at the biological process level 5, were performed with the DAVID bioinformatics database [16,17].

After 7 days in vitro (DIV) the medium was changed and neuron's RNA was extracted after 6, 24, and 72 hours. Microrarray analysis of gene expression in primary neurons showed that between the 6 and 24 hours after medium change 2001 genes appeared up-regulated while another 505 genes were down-regulated (Figure 1A). Following these changes in gene expression, analysis of the changes observed between 24 hours and 72 hours post-medium change indicated that 1325 genes were up-regulated between these two time points, while another 2579 were repressed (Figure 1B). Finally when comparing the gene expression profiles between 6 and 72 hours, 899 and 893 genes appeared induced and repressed respectively (Figure 1C).

Figure 1: Profile plot of significantly up- and down-regulated genes after medium change. Lines represent average expression profiles for significantly altered transcripts plotted against time. Changes in transcription between different times were identified by anova with p<0.05 using a fold change cut-off ≥2.

Analysis of the gene expression profile in relation to time in culture was then performed. A Venn diagram (Figure 2A) for up regulated genes in the three times assayed, showed that only 33 genes increased their expression starting at 6 hours and progressively increasing expression up to 72 hours (Figure 2B). Gene ontology analysis pointed that these genes were mainly involved in biological processes related to the immune response (Figure 2C and Supplementary Table 1). Another 402 genes were induced between the 6 and 72 hours after medium change with no change observed after 24 hours (Figure 2D). The majority of these genes could not be assigned to significant biological processes after gene ontology analysis. The remaining genes are involved in development, ion transport, adhesion, sensory perception, and regulation of signal transduction (Figure 2E). Finally, similarly to the previous case, the 250 genes with up-regulated expression after 24 hours, remained in this state after 72 hours (Figure 2F) and could not be assigned to any biological process. Those assigned are involved in the immune response (Figure 2G). Venn diagram was also performed for repressed genes (Figure 3A). This showed that the expression of only 8 genes progressively decreased between 6,24 and 72 hours (Figure 3B). No significant biological process involving these genes was identified after ontology analysis. Genes repressed during the first 24 hours after medium change, which remained in this state until 72 hours, also produced no significant biological process after ontology analysis (Figure 3C). The repression observed after 24 hours of medium change involved much more genes (Figure 3D), which are involved in biosynthesis/metabolism, development, transcription regulation, regulation of cell proliferation, response to unfolded protein and neuron differentiation (Figure 3E).

Figure 2: (A) Venn diagram for genes up-regulated between 6-24 hours, 24-72 hours, and 6-72 hours. (B) Box whisker plot of transcripts which progressively increased between 6 and 72 hours. (C) Biological processes significantly up-regulated between 6 and 72 hours identified using the DAVID database (Supplementary Table 1 for detailed information). (D) Box whisker plot of genes with increased expression between 24 and 72 hours after medium change. (E) Biological processes significantly up-regulated between 24 and 72 hours identified using the DAVID database (See supplementary Table 1 for detailed information). (F) Box whisker plot of genes with increased expression between 6 and 24 that remain increased up to 72 hours after medium change. (G) Biological processes significantly up-regulated between 24 and 6 hours that remain increased after 72 hours identified using the DAVID database (Supplementary Table 1 for detailed information).

Figure 3: (A) Venn diagram for genes down-regulated between 6-24 hours, 24-72 hours, and 6-72 hours. (B) Box whisker plot of transcripts which progressively decreased between 6 and 72 hours. (C) Box whisker plot of genes with decreased expression between 6 and 24 that remain repressed up to 72 hours after medium change. (D) Box whisker plot of genes with decreased expression between 24 and 72 hours after medium change. (E) Biological processes significantly down-regulated between 24 and 72 hours identified using the DAVID bioinformatic database (Supplementary Table 2 for detailed information).

To analyze how medium change transiently affected gene expression at 24 hours that reversed with time in culture at 72 hours, a clustering analysis was performed. After 72 hours in culture, most of the DE genes in the first 24 hours after medium change reverted their expression profile (Figure 4). Expression analysis showed that 1596 genes were induced in the first 24 hours after medium change but returned to their initial state at 72 hours (Figure 5A). Another 345 genes appeared repressed after 24 hours of medium change, increasing their expression between 24 and 72 hours (Figure 5B). Ontology analysis showed that transiently induced genes are involved in positive regulation of cell proliferation, immune response, biosynthesis/metabolism, and positive regulation of transcription. Transiently repressed genes are involved in the RHO/RAS signal transduction, and ion transport. Among these, several are important mediators/regulators of neuron function and development (Table 1).

Figure 4: Heat map obtained after hierarchical clustering of genes up- or down-regulated between 6 and 24 hours after medium change. Clusters of genes that are increased or decreased after 24 hours that revert the respective state after 72 hours are identified.

Figure 5: (A) Gene expression profile of induced genes after 24 hours of medium change that recover the expression the expression level observed after 6 hours. (B) Gene expression profile of repressed genes after 24 hours of medium change that recover the expression the expression level observed after 6 hours. (C) Biological processes significantly up- and down-regulated for identified genes, identified using the DAVID database (Supplementary Table 2 for detailed information).

Table 1: Significantly down-regulated genes after 24 hs of medium change (8 DIVs).

The results presented in this work show that medium renewal induced an important change in gene expression in primary neurons, which are incubated for several days in culture to achieve cell differentiation. In the first 24 hours after medium change, the repression and induction of hundreds of genes is observed, many of which recover to the initial state after 72 hours. This implies that gene expression alteration is a direct consequence of medium change and not of the cell differentiation that is in progress. Among the 33 genes that progressively increased their expression since medium change and up to 72 hours, are several involved in the immune response (CD38, PF4, FCGR2B, TGM2, C1QA, C1QB, C1QC). Of note is the progressive increase in the expression of C1QA, C1QB, C1QC, which code for the complement protein C1Q protein, formed by three subunits (C1Qα, C1Qβ, and C1Qγ) [18]. This protein, which is involved in synapse elimination and is a critical mediator of synaptic refinement and plasticity, has been shown to increase in aged mouse brains [19]. The increase of this protein reported in this work, evidence that primary neurons in culture, in the conditions described in this work, gradually differentiate with incubation time resembling normal neurons ageing. This protein is also considered neuroprotective since it increases neuronal survival and neurite outgrowth in the presence of β-amyloid induced neurotoxicity [20], and also triggers a gene expression program that enhances neurite outgrowth limiting the neuronal stress in vitro [21].

Several ionic channels, involved in specific neuronal functions, showed an increased expression after 10 DIVS when compared to the expression observed after 8 and 7 DIVs. This is probably a consequence of neurons differentiation and is not affected by medium change, since no effect was observed after 6 and 24 hours of medium renewal.

On the other hand, clustering analysis showed that the expression of several genes was significantly altered 24 hours after medium change (8 DIVs) but reverted this profile 48 hours latter (10 DIVs). Transiently down-regulated genes were identified to be involved in ion transport, transcription regulation, cell adhesion, and differentiation. Among ion channels affected were the potassium voltage-gated channels KCNC1 (Kv3.1), KCNG2 (Kv6.2), and KCNA4 (Kv1.4). The Kv3.1 channel subtype, that is widely distributed in the central nervous system playing a pivotal role in high-frequency repetitive firing of mammalian neurons [22,23], has been shown to be involved in neuronal differentiation [24], while the Kv1.4 channel subtype has been shown to be a component of the rapidly activating and rapidly inactivating voltage-gated A-type K+ current, IA, which acts regulating intrinsic excitability of cortical neurons [25]. Another channel subtype transiently down-regulated was SCN8A, which encodes sodium channel Nav1.6, one of the major voltage-gated sodium channels responsible for the generation and propagation of action potentials in mammalian neurons. It is present in the dendrites and soma of most neurons in the central and peripheral nervous systems [26,27]. In glutamatergic neurons this channel is strongly associated to the axon initial segment [28-31]. Besides the previously described channels, chloride channels and acid sensing channels also appeared down-regulated. These changes point to de differentiation of neurons. This notion is reinforced by the depression of the nucleotide exchange factor (GEFs) that activate the decrease in the expression of the small GTPases RHO/RAS. These GTPases are involved in neuronal development, through the regulation of actin and microtubule cytoskeleton [32]. We observed down-regulation of the GEFs ARHGEF15 (Ephexin5), which encodes a nucleotide exchange factor that negatively regulates synapse development limiting the number of excitatory synapses that neurons receive and, therefore, controlling synaptogenesis [33], ARHGEF28 (RGNEF), recently associated with the adult onset of amyotrophic lateral sclerosis [34], FGD2, an activator of Cdc42 (Rho GTPases family member), and of ARAP3, which inhibits RhoA activity leading to neurite outgrowth in response to nerve growth factor [35]. Other genes involved in neuron differentiation and morphogenesis (SEMA4C, CAPN3, WNT4, DPYSL5, VAX2, CBLN1) appeared also down-regulated again pointing to de-differentiation. These results imply that care should be taken in studies involving alterations in the above described genes and/or proteins since medium change could obscure or lead to misinterpretations of results.

Induced genes after 24 hours of medium change showed the expected response to renewal of nutrients and growth factors also pointing to partial loss of differentiation in cultured neurons. Among the genes induced are those involved in positive regulation of cell proliferation, biosynthesis/metabolism, and positive regulation of transcription all expected after medium renewal in cells in culture [36].

Taken together, the results indicate that in the time frame studied (7-10 days) a medium change at day 7 in culture induces a partial and transient de-differentiation that is reversed with time in culture as seen at day 10. In the same interval, several markers characteristic of differentiated neurons progressively increase from day 7 to day 10 with no interference observed after medium change.

The research work has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministry of Economy, Industry and Competitiveness, AGL2014-58210-R, AGL2016-78728-R, PI16/01830 and RTC-2016-5507-2. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD and ITC-20161072. From the European Union’s Seventh Framework Programme managed by REA-Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA